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Transgenic expression of epidermal growth factor and keratinocyte
growth factor in â-cells results in substantial morphological changes
M L Krakowski, M R Kritzik, E M Jones, T Krahl, J Lee,
M Arnush, D Gu, B Mroczkowski and N Sarvetnick
Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA
(Requests for offprints should be addressed to N Sarvetnick)
Abstract
The upregulation of a limited number of growth factors in
our interferon-ãtransgenic model for regeneration within
the pancreas lead us to propose that these factors are
important during pancreatic regeneration. In this study,
we have assessed the influence of two growth factors
within the pancreas, epidermal growth factor (EGF) and
keratinocyte growth factor (KGF), by ectopically express-
ing these proteins under the control of the human insulin
promoter in transgenic mice. This â-cell-targeted expres-
sion of either EGF or KGF resulted in significant
morphological changes, including cellular proliferation and
disorganized islet growth. Intercrossing the individual
Ins-EGF and Ins-KGF transgenic mice resulted in more
profound changes in pancreatic morphology including
proliferation of pancreatic cells and extensive intra-islet
fibrosis. Insulin-producing â-cells were found in some of
the ducts of older Ins-EGF and Ins-EGFKGF trans-
genic mice, and amylase-producing cells were observed
within the islet structures of the double transgenic mice.
These data suggest that both EGF and KGF are capable of
affecting pancreatic differentiation and growth, and that
co-expression of these molecules in islets has a more
substantial impact on the pancreas than does expression of
either growth factor alone.
Journal of Endocrinology (1999) 162, 167–175
Introduction
The healthy adult pancreas is a developmentally stable
organ with limited mitotic activity. Models allowing the
study of pancreatic growth and differentiation are of
particular interest because the elimination or dysfunction
of pancreatic cells is associated with a number of disease
states. In the human autoimmune disease insulin-
dependent diabetes mellitus, there is selective and
permanent destruction of the insulin-producing â-cells in
the islets of Langerhans. Pancreatic disorders also affect the
duct system, as is observed in chronic pancreatitis and
cystic fibrosis (Lack 1989, Sessa et al. 1990, Hootman & de
Ondarza 1993). It appears from these examples of disease
that the lost cells are not restored in vivo. Nevertheless, the
potential for such cells to regenerate has been shown
experimentally (Sarvetnick et al. 1988, Brockenbrough
et al. 1988, Rosenberg & Vinik 1989, Bonner-Weir et al.
1993, Wang et al. 1993, Wang & Bouwens 1995). We
have shown that the ectopic expression of interferon-ã
(IFN-ã) in the mouse results in regeneration within the
pancreas (Gu & Sarvetnick 1993). Further investigation
revealed that the expression of the growth factor epidermal
growth factor (EGF) was upregulated in the islets of these
transgenic mice (Arnush et al. 1996).
The growth factors required for pancreatic differentia-
tion are not yet fully defined. A survey of potentially
critical growth factors, including fibroblast growth
factor-2, recombinant human hepatocyte growth factor,
insulin-like growth factor-II, platelet-derived growth fac-
tor, and nerve growth factor did not identify any factors
that could influence the growth of pancreatic bud cultures
in vitro. However, EGF and transforming growth factor
â-1 (TGFâ-1) were able to induce ductal and endocrine
cell development respectively (Sanvito et al. 1994). While
the ability of EGF to stimulate epithelial cell and fibroblast
proliferation is well documented, it has also demonstrated
mitogenic properties for pancreatic growth (Dembinski
et al. 1982, Logsdon 1986, 1987, Marti et al. 1989, Verme
& Hootman 1990, Mangino et al. 1992). In addition,
evidence exists linking the overexpression of EGF and its
receptor to both chronic pancreatitis and malignant pan-
creatic growth (Barton et al. 1991, Korc et al. 1992, 1994,
Yamanaka et al. 1993, Friess et al. 1996). Indeed, over-
expression of this growth factor could potentially confer a
direct growth advantage to pancreatic cancer cells (Korc
1998).
The highly related member of the fibroblast growth
factor family, keratinocyte growth factor (KGF), has also
been shown to stimulate the proliferation of pancreatic
167
Journal of Endocrinology (1999) 162, 167–175
0022–0795/99/0162–167 1999 Society for Endocrinology Printed in Great Britain
Online version via http://www.endocrinology.org
ductal epithelial cells in rats after daily systemic injection
for 1–2 weeks (Yi et al. 1994). KGF is also involved in
wound healing (Werner et al. 1992) and in the differen-
tiation of many epithelial tissues (Aaronson et al. 1990,
Alarid et al. 1994). Additionally, KGF is known to
profoundly upregulate epidermal cell proliferation
(Aaronson et al. 1991). More specifically, we have pre-
viously observed that ectopic expression of KGF under the
control of the insulin promoter initiated significant intra-
islet proliferation of ductal cells in the pancreata of
transgenic mice (Krakowski et al. 1999).
For our IFN-ãtransgenic model, a screen for factors
potentially involved in the regenerating phenotype yielded
both EGF and KGF. Therefore, we chose to study how
these two factors might be involved by generating trans-
genic mice. In this study, we have investigated the
influence of these growth factors on pancreatic differen-
tiation by generating transgenic mice which overexpress
both EGF and KGF in the â-cells of the islets of
Langerhans. Here we report that co-expression of these
growth factors acted in concert to produce striking mor-
phological changes in the pancreas, without causing any
apparent abnormalities in pancreatic function.
Materials and Methods
Transgenic mouse generation
The 280 bp EGF cDNA was used to generate the
Ins-EGF transgenic mouse. The cDNA was cloned into a
vector containing the human insulin promoter and the
hepatitis B 3-untranslated sequence. The Ins-EGF frag-
ment was isolated by low melt agarose, purified using
Geneclean (BIO101 Inc., La Jolla, CA, USA) and NACS
Prepac DNA purification columns (BRL, Gaithersburg,
MD, USA), and microinjected into fertilized zygotes from
(BALB/cC57BL/6) F
2
mice. Progeny were screened
for the presence of the transgene by PCR typing of tail
DNA that was extracted using proteinase K digestion
overnight. PCR was performed using two 24-mer primers
specific for the human insulin promoter and EGF se-
quence. Transgene-positive mice were further bred with
BALB/c mice. The generation of Ins-KGF mice was
analogous and has been described elsewhere (Krakowski
et al. 1999). Once both lines had been backcrossed to
BALB/c mice for four generations, individual homozygote
Ins-EGF and Ins-KGF mice were mated and progeny
interbred to create homozygous, double transgenic
(Ins-EGFIns-KGF) mice.
Animal husbandry
All mice were maintained in a specific pathogen-free
facility at the Scripps Research Institute according to the
rules and regulations of the Institutional Animal Care and
Use Committee. Mice were housed under a controlled
12 h light: 12 h darkness cycle and provided with food and
water ad libitum.
Histological analysis and immunocytochemistry
Pancreata were fixed overnight in 10% neutral buffered
formalin (3·6% formaldehyde) and embedded in paraffin.
Paraffin sections (5 µm) were either conventionally stained
with hematoxylin and eosin (H&E) for histological evalu-
ation or stained for the presence of insulin, glucagon,
somatostatin, amylase, EGF, EGF receptor or bromo-
deoxyuridine (BrdU) using immunocytochemical tech-
niques (see Gu & Sarvetnick 1993, Arnush et al. 1996).
Sections were deparaffinized and blocked with 10% nor-
mal goat serum before applying the primary antibodies for
insulin, glucagon, amylase or somatostatin (all from
DAKO, Carpentaria, CA, USA), or BrdU (Accurate/
Sera-Lab, Westbury, NY, USA). Binding of the primary
antibody was detected using the appropriate secondary
antibody (Vector Laboratories, Burlingame, CA, USA or
Boehringer-Mannheim, Indianapolis, IN, USA), and the
horseradish peroxidase (HRP)-labeled avidin-biotin
complex (ABC kit; Vector Laboratories). HRP was
visualized using 3,3-diaminobenzidine as a substrate.
Gill’s hematoxylin was used as a counterstain. Masson’s
trichrome staining was completed at the Scripps Research
Institute Department of Histology. Briefly, paraffin-
embedded sections were fixed in Bouin’s fixative, stained
successively with Weigert’s iron hematoxylin and with
Biebrich scarlet-acid fuchsin acid then counterstained with
aniline blue. This results in collagen and mucin staining
blue indicating the presence of fibrosis while cytoplasm
stains red. Islet size was measured as in Krakowski et al.
(1999). Briefly, anti-insulin-stained sections from mice
of various ages were examined at 10power (Zeiss
Axioscope), using the 100 µm size of the crosshairs
for comparison. Islets were scored as small (<100 µm),
medium (200–400 µm) or large (>400 µm). Sixteen
Ins-KGF, sixteen Ins-EGF, eleven Ins-EGFKGF and
twelve non-transgenic mice, including all ages, were
examined. Statistical analyses of both young (<3 months)
and old (>3 months) mice for mean islet size (...)
were compared using the program Statview as described
below.
Blood glucose measurement
At regular intervals (approximately every 2 weeks), blood
was obtained from the tail and blood glucose levels were
determined using Glucofilm blood glucose test strips
(Miles Diagnostic, Elkhart, IN, USA). Non-fasting blood
glucose values of non-transgenic BALB/c mice in our
colony ranged from 80 to 160 mg/dl.
M L KRAKOWSKI and others · Expression of EGF and KGF in the pancreas168
Journal of Endocrinology (1999) 162, 167–175
Glucose tolerance testing
To determine glucose tolerance over time, transgenic and
control littermate mice were fasted overnight and an initial
blood glucose reading taken (time zero) as described
above. Mice were then injected intraperitoneally with
0·2 g -glucose/100 g body weight (0·1 g/ml in sterile
PBS), and blood glucose measured after 10 min and again
after 60 min.
BrdU labeling
Cellular proliferation was quantified by injecting 100 µg/g
body weight BrdU (Serva, Heidelberg, Germany) intra-
peritoneally into mice 15 h prior to their being killed.
Paraffin-embedded pancreata were sectioned and stained
with an anti-BrdU antibody (Accurate Chemical,
Westbury, NY, USA) as described above after treatment
with 2·8 M HCl for 15 min. The extent of proliferation
was determined by counting all the BrdU-positive cells
within 20 fields using a 20objective; the area equiva-
lent to one field at this magnification is exactly 4·010
5
µm
2
. We specifically made note of whether the positive
cell was a duct cell or found within the islet of Langerhans
and/or acinar tissue. This measurement of 20 fields was
representative of all the BrdU-positive cells per pancreatic
slice, yet was also normalized for the area measured
for each mouse. Six Ins-KGF, five Ins-EGF, eight
Ins-KGFEGF and six non-transgenic mice were
counted. This allowed the quantification of the average
number of BrdU-positive cells (...) per pancreatic
slice and statistical analyses.
Statistical analysis
The analysis of variance (unpaired t-test) test was used to
compare differences between the groups using the
Statview program by Abacus Concepts (Berkeley, CA,
USA).
Results
Generation of Ins-EGF transgenic mice
To determine the influence of EGF expression in the
â-cells of the pancreas, transgenic mice expressing murine
EGF under control of the human insulin promoter were
generated (Ins-EGF). Two lines of transgene-positive
mice were found when screened by PCR and were
further bred with BALB/c mice for four generations from
the original (BALB/cC57BL/6) F
2
mice. No signifi-
cant differences between the lines were ever observed.
These transgenic mice appeared normal and healthy, with
average life spans. We characterized expression patterns of
EGF by immunohistochemistry. The expression of EGF
was found to be significantly upregulated in all transgenic
islets (Fig. 1A). As expected, EGF was expressed at low
levels in the islets of non-transgenic littermate controls
(Fig. 1B). Furthermore, while the level of the EGF
receptor (EGF-R) appeared comparable to the low con-
stitutive level of expression previously observed for this
receptor (Damjanov et al. 1986, Chabot et al. 1987), we
did detect a modest upregulation of the EGF-R in the
ductal epithelia of the transgenic pancreas (data not
shown).
Morphological changes of Ins-EGF mice
Ins-EGF transgenic mice had dramatic morphological
changes within their pancreata. While the pancreatic ducts
appeared normal in the transgenic mice, the islets were
found to increase in size as the mice aged. After 3 months
of age, these mice had significantly more islets of larger size
(>400 µm in diameter) than did their non-transgenic
littermates, in which most islets were <100 µm in
diameter (P<0·008). In addition, the transgenic islets
exhibited substantial disaggregation (Fig. 1). We also
observed significant fibrosis around the islets (Fig. 1D).
Pancreatic lymphocytic infiltration was extremely minor
and not sufficient to explain the disorganized architecture
of the islets. Surprisingly, the rare lymphocyte infiltration
we first observed at 3 months did not increase to any
significant level with age (Fig. 1C: Ins-EGF, 3 months and
Fig. 1D: Ins-EGF, 17 months). The structural changes in
the islets were not observed in non-transgenic littermates
(Fig. 1E).
Immunostaining for insulin, glucagon, somatostatin,
and amylase in the Ins-EGF mouse revealed typical
expression patterns within the islets and exocrine tissue,
similar to age- and sex-matched non-transgenic controls
(Fig. 2C and D and data not shown for hormones other
than insulin). Additionally, the cells producing these
proteins were, for the most part, present in typical
numbers and placement. However, we did detect the rare
presence of insulin-positive cells in some ducts at a later
age (Fig. 3A), implying the generation of newly formed
â-cells within the pancreatic ducts; â-cells are not
normally found outside of the islets. In spite of the unusual
islet morphology, the overall health and viability of
Ins-EGF mice matched that of non-transgenic littermates.
Blood glucose values were taken at 2-week intervals for
mice of various ages and no mouse exceeded the normo-
glycemic value of 150 mg/dl, nor was any mouse found to
be hypoglycemic.
Synergistic effects of combined EGF and KGF on pancreatic
morphology
We have previously generated a model of intra-islet duct
cell proliferation by ectopic expression of KGF under
control of the insulin promoter (Krakowski et al. 1999).
The expression of KGF in the islets of the pancreas
Expression of EGF and KGF in the pancreas ·M L KRAKOWSKI and others 169
Journal of Endocrinology (1999) 162, 167–175
resulted in enlarged islets, with substantial proliferation of
duct cells within the islet mass. In addition, we observed
the presence of hepatocyte-like (albumin and á-
fetoprotein-producing) cells in the islets of the KGF-
expressing transgenic mice (Krakowski et al. 1999). These
pancreatic heptatocytes are easily recognizable as the
non-insulin-producing cells that are extremely large in size
compared with the other islet cells. Additionally, they are
found on the periphery of the islet and are often binucleate
(Fig. 2F, see arrowheads). Despite the morphological
differences in the pancreata of KGF transgenic mice, no
pathology, hyperglycemia, or hypoglycemia were found to
be associated with KGF expression in the islets of trans-
genic mice. To determine the effects that â-cell expression
of these related growth factors would have on pancreatic
differentiation and function, Ins-EGF mice were crossed
to Ins-KGF mice, and Ins-EGFKGF progeny were
interbred to homozygosity for both transgenes. Charac-
terization of these double transgenic mice revealed unusual
changes in pancreatic morphology which did not simply
reflect the effects of each individual transgene.
Ins-EGFKGF mice developed enlarged, distended,
and non-confluent islets at earlier ages than was seen for
either the single Ins-EGF or Ins-KGF transgenic mice.
Additionally, all of the transgenic mice, aged 3 months or
greater, had significantly more islets of larger size
(>400 µm diameter) (Fig. 2A–F) than did non-transgenic
mice (Fig. 2G and H), in which most islets were <100 µm
(P<0·02). The mean of the number of large islets per
pancreatic slice in mice aged 3 months or greater was:
Figure 1 Overexpression of EGF within islets of transgenic Ins-EGF mice leads to disorganization of islet architecture and increases with
age. Three-month-old Ins-EGF (A) and age- and sex-matched non-transgenic control littermate (B) both stained with anti-EGF antibody as
shown in brown chromagen staining against blue hematoxylin counterstain. Three-month-old (C) and 17-month-old (D) Ins-EGF mice
stained with H&E, revealing rare and sparse mononuclear cell infiltration to the irregularly shaped islets that increased in size and
complexity with age. Age- and sex-matched non-transgenic control littermate stained with H&E (E), demonstrating typical islet morphology
and no immune infiltration. Original magnification of A: 20andB:32, C–E: 20 .
M L KRAKOWSKI and others · Expression of EGF and KGF in the pancreas170
Journal of Endocrinology (1999) 162, 167–175
Figure 2 Morphological changes occur at an earlier age in Ins-EGF KGF mice, and are more severe than
in single transgenic mice. Three-month-old (A) and 6-month-old (B) Ins-EGFKGF mice stained with
anti-insulin antibody (shown in brown) and counterstained with blue hematoxylin showing increasingly
larger, and more disorganized networks of islets. Compare the effects of increasing age from 3 (left panel)
to 6 months (right panel) on islet of Langerhans morphology in C, D: Ins-EGF, E, F: Ins-KGF and G, H:
non-transgenic (all age-matched) demonstrating delayed and lesser phenotypic changes in single transgenic
mice and normal morphology in non-transgenic controls. Arrowheads in panel F indicate pancreatic
hepatocytes within lns-KGF mouse. Original magnification for all photos was 20.
Expression of EGF and KGF in the pancreas ·M L KRAKOWSKI and others 171
Journal of Endocrinology (1999) 162, 167–175
Ins-KGF 2·70·6 (n=8), Ins-EGF 3·30·6 (n=13),
Ins-EGFKGF 5·50·7 (n=4) and non-transgenic
0·90·4 (n=8). In this regard, the double transgenic mice
(Fig. 2A and B) had more islets of larger size than did the
Ins-KGF transgenic mice (P<0·02) (Fig. 2E–F), and there
appeared to be more islets of a larger size in the double
transgenic mice than in the Ins-EGF mice (Fig. 2C and
D), although the difference was not as significant
(P<0·07). Morphological changes to the islets continued
to develop as the mice aged. Islet and ductule networks
were much larger in the double transgenic mice than in
either single transgenic mouse. Furthermore, there ap-
peared to be an increase in the number of intra-islet
ductules (a representative intra-islet duct is shown in
Fig. 3B). Although islets were enlarged, distended, and
lobulated to varying degrees for each transgenic mouse,
individual â-cells remained intact and functional as
demonstrated by insulin staining (Fig. 2). Staining for
the hormones somatostatin and glucagon revealed typical
patterns as seen in non-transgenic littermates (data not
shown). Interestingly, as seen in the Ins-EGF mouse,
insulin-positive cells were also observed in some of the
ducts of the Ins-EGFKGF transgenic mice at a later age
(6 months) (data not shown).
Normal, non-fasting blood glucose levels were
measured over the animal’s lifespan (90–150 mg/dl). Yet
the question remained whether these mice regulated their
insulin production normally in the face of such unusual
morphological changes. All three types of transgenic mice
and control mice were analysed with a glucose tolerance
test (Wogensen et al. 1993). We observed some inhibition
of blood glucose homeostasis immediately following
(10-min time-point) the injection of glucose, but after
60 min all mice were indistinguishable and had normal-
ized their blood glucose levels (Table 1). These data may
indicate an abnormality in the first phase of insulin release,
but no detriment was experienced by the mice as their
non-fasting blood glucose levels were normal over their
entire lifetimes.
To assess the effects of EGF and KGF on cell prolifer-
ation, mice were injected with BrdU; BrdU incorporation
into pancreatic cells was determined by immunohisto-
chemistry. The extent of proliferation was determined by
counting the number of BrdU-positive cells in 20 fields
Figure 3 Ins-EGF KGF mice have intra-islet ducts, ductal insulin-positive cells, fibrosis and amylase-positive cells within islets of
Langerhans. Seventeen-month-old female Ins-EGF mouse stained as in Fig. 2 shows insulin-producing cells within the duct wall (A).
Intra-islet duct (designated D’) identified by CAII staining in a 5·5-month-old male mouse (B). Trichrome staining reveals a severe degree
of fibrosis within the Ins-EGFKGF transgenic mice (4-month-old male) (D) in contrast to the non-transgenic age- and sex-matched
littermate (C). Fibrosis within the islets appears as bright blue spindle shaped cells with elongated nuclei, and increases with age (data not
shown). Anti-amylase antibody stained with brown chromogen against a blue hematoxylin counterstain reveals atypical endocrine as well
as the expected exocrine staining within the pancreata of Ins-EGFKGF mice. (E) Early stages of engulfment of acinar tissue by ongoing
fibrotic reaction occurring around the islet. (F) Acinar cells staining positive for amylase surrounded by islet tissue and fibrotic cells. (G)
Amylase-positive cells without distinct acinar cell morphology within islet tissue. Original magnification of A: 40,B:20,CandD:
40,E:20and F and G: 40 .
M L KRAKOWSKI and others · Expression of EGF and KGF in the pancreas172
Journal of Endocrinology (1999) 162, 167–175
per section at 20magnification. This measurement was
representative of the number of BrdU-positive cells per
pancreatic slice. On average, we found that Ins-EGF and
Ins-KGF mice had 6·82·0 (n=5 mice) and 11·03·8
(n=6 mice) BrdU-positive cells respectively. In contrast,
the double transgenic mice had 15·33·1 (n=8 mice) and
the non-transgenic littermates had 0·70·3 (n=6 mice)
positive cells. Thus all three lines of transgenic mice had
significantly more pancreatic cell proliferation than did the
control mice (P<0·01). The majority of the proliferating
cells of the Ins-KGF mouse had been previously identified
as ductal (Krakowski et al. 1999). Morphological exami-
nation and statistical comparison (t-test completed for each
transgenic mouse), indicated that proliferating cells of the
Ins-EGF and of the Ins-EGFKGF mice were found to
be distributed without bias in the acinar and islet, versus
ductal tissues (Pvalue varied between 0·13 and 0·46,
indicating no significant difference).
As with the Ins-EGF mice, pancreatic lymphocytic
infiltration was rare and sparse and did not increase with
age in the double transgenic Ins-EGFKGF mouse (data
not shown). Immunohistochemistry was not completed to
characterize the cellular composition of the infiltrates for
any of the transgenic mice because of the rarity and
subsequent presumed lack of clinical significance the
immune cells had for the phenotype. Interestingly, much
more extensive intra-islet fibrosis was observed in the
double transgenic mice than was observed in the Ins-EGF
mice (Fig. 3C and D). As seen in this Figure, the fibrosis
is characterized by spindle shaped cells with elongated
nuclei. The level of fibrosis increased substantially with
age in the double transgenic mice. It is highly probable
that the exogenously expressed growth factors are acting,
perhaps indirectly, on fibroblasts and other cells of epi-
thelial origin to stimulate this fibrosis. Evidence to support
a role for the involvement of growth factors in fibrosis
comes from the TGFâmouse (Sanvito et al. 1994) as well
as those described here.
Unexpectedly, we detected the presence of exocrine
cells, which stain positive for amylase, within the islets of
double transgenic mice; such positive staining was not
observed within the islets of either single transgenic mouse
or non-transgenic control. Figure 3E–G illustrates the
morphology of amylase-positive islet sections, in what we
believe to represent progressive stages in the evolution of
this phenomenon. That is, it appears that amylase-positive
cells are engulfed by the ongoing fibrotic reaction occur-
ring adjacent to islets (Fig. 3E-G), perhaps due to dis-
rupted matrix connections or altered adhesion between
cells, after which the membranes of the engulfed cells
break down to leave a residual fragment of exocrine tissue,
which stains positive for amylase (Fig. 3G). The presence
of these exocrine cells within the endocrine (islet) tissue is
at least partially responsible for the non-confluent insulin
staining we observed in the islets of double transgenic
mice, as the cells which produce amylase likely displace
the insulin-producing cells.
Discussion
We have generated a model system in which the expres-
sion of KGF and EGF has been targeted to the â-cells in
the islets of Langerhans. KGF and EGF are members of a
large family of very similar growth factors. Members of this
family have been shown to influence a number of pro-
cesses, including cell proliferation, migration, and differ-
entiation (Brown 1995). We sought to determine the
influence of these molecules on pancreatic development
because previous studies have suggested that these
molecules might play important roles in this process. For
example, previous work by Yi et al. (1994) demonstrated
that systemic administration of KGF to rats induced
pancreatic duct cell proliferation. The intralobular duct
cell proliferation observed was predominantly adjacent to
or within the islets of Langerhans, and occurred in the
absence of physical injury to the pancreas (Yi et al. 1994).
In addition, while investigating the remarkable prolifera-
tive and differentiation patterns of the Ins-IFN-ãmouse,
EGF and EGF-R were found to be upregulated (Arnush
et al. 1996). Indeed, EGF has previously been shown to
modulate pancreatic growth (Dembinski et al. 1982,
Logsdon 1986, 1987, Marti et al. 1989, Verme & Hootman
1990, Mangino et al. 1992). For example, systemic admin-
istration of EGF resulted in the induction of pancreatic
duct proliferation in pigs (Vinter-Jensen et al. 1997).
While the Ins-EGFKGF transgenic mouse enabled us
to address the co-operative effects that localized over-
production of these molecules has on pancreatic growth
and function, the Ins-EGF and Ins-KGF (described in a
separate manuscript; Krakowski et al. 1999) transgenic
mice enabled us to assess their influences independently.
Although a low basal level of EGF is normally expressed
in the islets of non-transgenic mice, KGF is not normally
found at this location. Our transgenic mouse models have
demonstrated that expression of EGF and KGF in islet
Table 1 Typical pattern of fluctuations in blood glucose. Values are
meansS.E.M.
Blood glucose (mg/dl)
Transgene 0 min 10 min 60 min
Ins-KGF (n=3) 673 296 94 11629
Ins-EGFKGF (n=4) 119 13 348105 12216
Ins-EGF (n=3) 1087 466 32 141109
BALB/c (n=4) 10913 205 26 12812
Fasting blood glucose concentration (mg/dl) in all three transgenic and
control mice during an intraperitoneal glucose tolerance test of male
mice of various ages (range between 3 and 14·5 months) Data are given
as the mean and standard error for each type of transgenic. Blood for
determination of the blood glucose was taken from the retro-orbital venous
plexus during anaesthesia.
Expression of EGF and KGF in the pancreas ·M L KRAKOWSKI and others 173
Journal of Endocrinology (1999) 162, 167–175
â-cells generates an accelerated and extensive series of
changes to both endocrine and exocrine tissues. For
example, we have observed significant intra-islet duct cell
proliferation in the pancreata of Ins-KGF mice. In
addition, we have found that hepatocyte cells exist within
the islets of these transgenic mice. Pancreatic cell prolifer-
ation was also observed in the Ins-EGF and Ins-EGF
KGF mice. The Ins-EGF and Ins-EGFKGF transgenic
mice also exhibited disorganized islets and intra-islet
fibrosis as well, both of which were more extensive in the
double transgenic mouse. We also found that many
features shared by both single transgenic mice, such as
pancreatic cell proliferation, increased size and disorgan-
ization of islets, and fibrosis, were seen to a greater extent
in the double transgenic mice. Interestingly, the amylase-
positive cells seen in the double transgenic mice are not
found in either of the single transgenic mice, indicating
that localized overexpression of both EGF and KGF in
â-cells is required to produce this unique phenotype.
Despite the extensive morphological changes that are
observed in the pancreata of growth factor transgenic
mice, only very minor deficiencies in pancreatic function
were detected. All of the endocrine hormones and exo-
crine enzymes were present in these transgenic mice, and
blood glucose levels remained normal throughout the lives
of the animals. Thus the physical changes apparent in these
mice did not interfere with normal pancreatic function.
Interestingly, some of the pathologies observed also char-
acterize several pancreatic diseases. For example, the GK
rat model of non-insulin-dependent diabetes is character-
ized by disorganized islets, significant fibrosis, and clusters
of â-cells separated by strands of connective tissue
(Movassat et al. 1995). In addition, chronic pancreatitis is
characterized by inflammation and fibrosis (Steer 1989).
Interestingly, in chronic pancreatitis and in some human
pancreatic cancers, EGF, EGF-R, and KGF are often
overexpressed (Barton et al. 1991, Korc et al. 1992, 1994,
Yamanaka et al. 1993, Siddiqi et al. 1995, Friess et al.
1996). These observations suggest that these factors might
play a role in disease progression and pathology. For
instance, it is possible that altered expression of these
critical growth modulators could confer a significant
growth advantage to pancreatic cancer cells.
The physiological roles of KGF and EGF in pancreatic
development are not clear. Indeed, KGF knockout mice
do not appear to have any abnormalities in pancreatic
development or function (Guo et al. 1996). However,
there is likely to be redundancy within the large family of
highly related proteins to which these proteins belong,
perhaps masking the contribution of individual factors
during development. Such influences would be expected
to become more apparent when overexpression of these
proteins is localized, perhaps manifest in the phenotypes
we observe. Indeed, as discussed earlier, these growth
factors do appear to influence pancreatic growth and
differentiation, in vivo and in vitro. The observation that
several uncharacteristic cell types exist in the transgenic
mice overexpressing EGF and KGF in islets also suggests a
possible role for these growth factors in pancreatic
differentiation. We have detected the presence of
insulin-producing cells in the ducts of the Ins-EGF and
Ins-EGFKGF transgenic mice and of hepatocyte-like
cells in the islets of KGF transgenic mice. Thus, EGF and
KGF might contribute to pancreatic differentiation by
promoting cellular lineage commitment along specific
pathways. However, as in any model system, it is possible
that the phenotypes exhibited by our transgenic mouse
models are due to targeted overexpression in islets and are
not reflective of innate physiological influences. Future
studies designed to assess the influence of these growth
factors on pancreatic growth and differentiation will
therefore be important in addressing these critical issues.
In summary, we have developed a transgenic mouse
system which will enable us to study the effects of EGF
and KGF on pancreatic growth, differentiation, and on the
pathologies associated with aberrant overexpression of
these growth factors. In addition, the transgenic mice we
have produced will enable us to study the generation
of the distinct cell types described here, such as the
hepatocyte-like cells in the Ins-KGF mice and the ductal-
endocrine cells in the Ins-EGF and Ins-EGFKGF mice.
As such, these studies will enhance our understanding of
how these critical growth modulators contribute to the
growth and differentiation of the pancreas.
Acknowledgements
The authors would like to heartily thank Gail Patson and
Augusta Good who maintained and screened the mouse
colony. We would also like to thank Margaret A Chadwell
for completing the Trichome stains. The administrative
assistance of Joanne Dodge and Jackie Soto is always
gratefully appreciated. Drs Marc Horwitz and Malin
Flodstrom are thanked for providing provocative scientific
discussion.
Grant numbers and sources of support: M L K is
supported by an NMSS postdoctoral fellowship,EMJwas
supported by NIH postdoctoral fellowship DK09355–01,
D G was supported by a postdoctoral fellowship from the
Juvenile Diabetes Foundation, andNSissupported by a
Diabetes Interdisciplinary Research Center from the
Juvenile Diabetes Foundation and by NIH grant
HD-29764 and JDFI 995010. This is publication
11669-IMM from the Department of Immunology, the
Scripps Research Institute.
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Received 12 January 1999
Accepted 23 March 1999
Expression of EGF and KGF in the pancreas ·M L KRAKOWSKI and others 175
Journal of Endocrinology (1999) 162, 167–175
